TSG-6 Inhibits Osteoclast Activity via an Autocrine Mechanismand Is Functionally Synergistic With Osteoprotegerin
David J. Mahoney,1 Catherine Swales,1 Nicholas A. Athanasou,1 Michele Bombardieri,2
Costantino Pitzalis,2 Karolina Kliskey,1 Mohammed Sharif,3 Anthony J. Day,4
Caroline M. Milner,4 and Afsie Sabokbar1
Objective. TSG-6 (the product of tumor necrosisfactor [TNF]–stimulated gene 6) has a potent inhibitoryeffect on RANKL-mediated bone erosion. The aim ofthis study was to compare the activity of TSG-6 withthat of osteoprotegerin (OPG) and to investigate its roleas an autocrine modulator of cytokine-mediated oste-oclast formation/activation. We also determined TSG-6expression in inflammatory joint disease.
Methods. The effects of TSG-6, OPG, and theinflammation mediators TNF�, interleukin-1 (IL-1),and IL-6 on the formation of osteoclasts from pe-ripheral blood mononuclear cells and synovial fluid(SF) macrophages were determined by tartrate-resistant acid phosphatase staining. Lacunar resorptionand filamentous actin ring formation were measured asindicators of osteoclast activity. The amount of TSG-6 inculture media or SF was quantified by enzyme-linked
immunosorbent assay, and expression of TSG-6 insynovial tissue was assessed by immunohistochemistry.
Results. TSG-6 acted in synergy with OPG toinhibit RANKL-mediated bone resorption and was pro-duced by osteoclast precursors and mature osteoclastsin response to TNF�, IL-1, and IL-6. Expression ofTSG-6 correlated with inhibition of lacunar resorption;this effect was ameliorated by an anti–TSG-6 antibody.The level of TSG-6 protein was determined in SF frompatients with various arthritides; it was highest inpatients with inflammatory conditions such as rheuma-toid arthritis, in which it correlated with the amount ofTSG-6 immunostaining in the synovium. TSG-6 inhib-ited the activation but not the formation of osteoclastsfrom SF macrophages.
Conclusion. In the presence of inflammatory cy-tokines, osteoclasts produced TSG-6 at concentrationsthat are sufficient to inhibit lacunar resorption. Thismay represent an autocrine mechanism to limit thedegree of bone erosion during joint inflammation.
TSG-6 (the protein product of tumor necrosisfactor [TNF]–stimulated gene 6) (1) is not constitutivelyexpressed in most tissues but is up-regulated in responseto proinflammatory mediators (2,3). It is composed ofcontiguous Link and CUB modules flanked byN-terminal and C-terminal peptides and binds to diverseligands that include bone morphogenetic proteins,RANKL, and glycosaminoglycans (e.g., hyaluronan andheparin) (2–7). TSG-6 expression is associated withinflammatory diseases such as arthritis, and it has beendetected in the synovial fluid, cartilage, and synovium ofpatients with osteoarthritis (OA) and patients with rheu-matoid arthritis (RA) but not in control subjects withoutarthritis (8,9). TSG-6 is also produced by culturedhuman chondrocytes in response to TNF�, interleukin-1(IL-1), IL-6, and transforming growth factor � (10,11),
Supported by Arthritis Research UK (grants 17590 to Drs.Day, Milner, and Sabokbar, 16539 to Drs. Day and Milner, 18358 toDr. Swales, 18399 to Drs. Bombardieri and Pitzalis, and 18237 to Dr.Bombardieri) and by Isis Innovation, Oxford. Dr. Athanasou’s workwas supported by grants from the Rosetrees Trust and the OxfordRegional Health Trust.
1David J. Mahoney, DPhil, Catherine Swales, PhD, MRCP,Nicholas A. Athanasou, MD, PhD, Karolina Kliskey, BSc, AfsieSabokbar, PhD: University of Oxford, Oxford, UK; 2Michele Bombar-dieri, MD, PhD, Costantino Pitzalis, MD, PhD, FRCP: Barts and theLondon School of Medicine and Dentistry, London, UK; 3MohammedSharif, PhD: University of Bristol, Bristol, UK; 4Anthony J. Day,DPhil, Caroline M. Milner, DPhil: University of Manchester, Man-chester, UK.
Address correspondence to Afsie Sabokbar, PhD, BotnarResearch Centre, University of Oxford, Windmill Road, Headington,Oxford, OX3 7LD, UK (e-mail: [email protected]);or to Anthony J. Day, DPhil, or to Caroline M. Milner, DPhil, Facultyof Life Sciences, University of Manchester, Michael Smith Building,Oxford Road, Manchester, M13 9PT, UK (e-mail: [email protected] or [email protected]).
Submitted for publication March 26, 2010; accepted in revisedform December 10, 2010.
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and its constitutive expression by RA synoviocytes isenhanced by IL-1 (8), TNF� (8), and IL-17 (12).
TSG-6 has been reported to have a protectiverole in experimental models of both arthritis (13–17) andacute inflammation (18–20). In collagen-induced arthri-tis, systemic treatment with recombinant human TSG-6(rhTSG-6) caused a delay in the onset of symptoms andreduced joint inflammation/destruction (14,17). Chon-droprotective effects were also observed when recombi-nant murine TSG-6 was administered to mice withantigen-induced arthritis or proteoglycan-induced ar-thritis (13,15). These antiinflammatory and chondropro-tective properties are likely to be attributable to morethan one mechanism, because TSG-6 has diverse bio-logic activities, including the inhibition of neutrophilmigration (16,18–20) and the down-regulation of plas-min activity (6,18,19).
Our group recently reported that TSG-6 also hasantiresorptive activity, whereby the full-length proteinand, to a lesser extent, its isolated Link module inhibitRANKL-induced osteoclastic bone erosion in vitro (5).TSG-6 binds directly to RANKL and inhibits boneresorption with a potency similar to that of osteoprote-gerin (OPG; a soluble decoy receptor for RANKL),suggesting that it might act via a mechanism similar tothat of OPG, i.e., by preventing RANK–RANKL inter-action (5). However, although OPG inhibits osteoclastformation and activation, TSG-6 has no apparent effecton the former (5). This study is the first to demonstratethat TSG-6 and OPG act synergistically to inhibit oste-oclastic bone erosion. We also provide evidence thatTSG-6 is secreted by osteoclast precursors and matureosteoclasts in response to proinflammatory cytokines,and that it acts in an autocrine manner to regulate boneerosion during inflammation.
PATIENTS AND METHODS
Patients and samples. Synovial fluid (SF) was aspi-rated from the knee joints of 19 patients with OA (7 men and12 women, average age 76 years, average disease duration 10years), 33 patients with RA (8 men and 25 women, average age60 years, average disease duration 11 years), 7 patients withpsoriatic arthritis (5 men and 2 women, average age 54 years,average disease duration 5 years), 10 patients with gout (9 menand 1 woman, average age 53 years, average disease duration 3years), and 9 patients with pyrophosphate arthritis (PPA; alsoknown as calcium pyrophosphate deposition disease or pseu-dogout) (all men, average age 76 years, average diseaseduration 10 years). The diagnoses were made based on clinical,radiographic, or histologic criteria. Cases of OA were classifiedas inflammatory (n � 11; 4 men and 7 women) or noninflam-matory (n � 8; 3 men and 5 women) according to the
proportion of neutrophils detected within the SF (21). SFsamples with neutrophils totaling �25% of the total cellpopulation were considered noninflammatory, while thosewith �80% neutrophils were defined as inflammatory; samplesin which the counts were between these 2 limits were notanalyzed. SF samples from individuals with no knee pain andno history or evidence of joint disease (1 man and 2 women,average age 33.3 years) were used as controls. Synovial biopsyspecimens were also obtained from 9 patients with RA. Ethicsapproval was obtained from the National Research EthicalCommittee, and patient consent was acquired prior to thecollection of samples.
Reagents. Full-length rhTSG-6 and the isolated Linkmodule of TSG-6 (Link_TSG6) were expressed and purified asdescribed previously (22–24). Recombinant human cytokineswere purchased from R&D Systems Europe, except for solubleRANKL (sRANKL), which was obtained from PeproTech. Arat monoclonal antibody (A38.1) raised against the Linkmodule of human TSG-6 was generated as described previ-ously (25). All tissue culture media and supplements werepurchased from Invitrogen.
Synergistic effects of TSG-6 and OPG on sRANKL-mediated osteoclast resorption. Human peripheral blood mono-nuclear cells (PBMCs) were isolated from healthy male volun-teers (ages 25–35 years) and seeded on dentine slices, asdescribed previously (5). Cells were subsequently incubated in1 ml of �-minimum essential medium (�-MEM) supplementedwith 10% (volume/volume) fetal calf serum (FCS), 10 mML-glutamine, antibiotics (100 IU/ml penicillin and 10 �g/mlstreptomycin), 25 ng/ml macrophage colony-stimulating factor(M-CSF), and 50 ng/ml sRANKL. OPG, rhTSG-6, and Link-_TSG6 were added to the media, alone or together, at a rangeof concentrations. Cultures were maintained for 21 days,during which time the media containing all factors werereplaced approximately every 3 days. Resorption lacunae ondentine slices were then measured in quadruplicate for eachindividual, as described previously, as an indicator of osteoclastactivity.
Quantification of TSG-6 and OPG production duringosteoclastogenesis in vitro. In order to determine whetherosteoclast precursors or mature osteoclasts are capable ofsecreting TSG-6 or OPG in culture, human PBMCs wereseeded onto 6-well plates (4 � 106 cells/well) in �-MEMcontaining FCS and antibiotics, as described above. PBMCswere incubated with 25 ng/ml M-CSF in the presence of 50ng/ml IL-1, IL-6, TNF�, IL-17, LIGHT, or sRANKL; mediawere changed every 3–4 days. After 21 days, osteoclast forma-tion was assessed by determining the presence of multinucle-ated tartrate-resistant acid phosphatase (TRAP)–positive cells(5). In addition, TSG-6 in the tissue culture media wasquantified by enzyme-linked immunosorbent assay (ELISA),as described previously (26), with a minimum detectableconcentration of 1 ng/ml. Human OPG concentrations wereassessed using a DuoSet ELISA System (R&D Systems Eu-rope), with a minimum detection level of 0.1 ng/ml. TSG-6 wasalso quantified in the media collected from cells treated withIL-1, IL-6, or TNF� (0, 1, 5, 10, and 50 ng/ml) during thecourse of osteoclastogenesis (on day 3, 7, 10, 14, and 17) and atthe end point of the assay (day 21).
TSG-6 IS AN AUTOCRINE REGULATOR OF BONE RESORPTION 1035
Effect of cytokine supplementation on RANKL-induced osteoclast resorption. Human PBMCs were seededonto dentine slices (for lacunar resorption assays) or coverslips(for determination of cell viability) and cultured in the pres-ence of M-CSF and sRANKL, as described previously (5) andas outlined above. Wells were supplemented with 0, 1, 5, 25, or75 ng/ml of TNF�, IL-1, or IL-6, and the cultures weremaintained for 21 days. TSG-6 protein in the media from cellscultured on dentine slices was then quantified by ELISA, andthe area of resorption lacunae was determined. PBMCs cul-tured on coverslips were fixed with formalin, washed twice inphosphate buffered saline (PBS) to remove serum, and stainedusing a LIVE/DEAD viability assay kit according to themanufacturer’s instructions (Molecular Probes); the inclusionof 3 �M 4�,6-diamidino-2-phenylindole (Invitrogen) allowedvisualization of cell nuclei. Coverslips were rinsed twice inPBS, and the proportion of live-to-dead cells was determinedusing a Zeiss Axioplan microscope.
Effects of TSG-6 on osteoclast activation. The effect ofTSG-6 on filamentous actin (F-actin) ring formation (i.e., anindicator of attachment to the bone by active osteoclasts) wasassessed. PBMCs were seeded onto dentine slices and culturedas described above with M-CSF and sRANKL, in the presenceor absence of rhTSG-6 (250 ng/ml). After 21 days, adherentosteoclasts were fixed in 4% (v/v) paraformaldehyde, washedin PBS, and stained with tetramethylrhodamineisothiocyanate–conjugated phalloidin (500 ng/ml) for 45 min-utes. Dentine slices were thoroughly washed in PBS, mountedin FluorSave (Calbiochem), and examined with an OlympusBX40 microscope. The inhibitory effect of TSG-6 (produced in
response to TNF�) on osteoclast resorption was verified usinga TSG-6–specific monoclonal antibody (A38.1). PBMCs werecultured on dentine slices with M-CSF and sRANKL (asdescribed above, with media changes approximately every 3days) in the presence or absence of rhTSG-6 (250 ng/ml),TNF� (50 ng/ml), and A38.1 (1 �g/ml); the extent of lacunarresorption was measured after 21 days. Cells cultured withTNF� in the presence or absence of A38.1 (without sRANKLor M-CSF) were used as controls.
Immunohistochemical analysis of the synovium andbone–cartilage interface. Formalin-fixed, paraffin-embeddedarthroplasty and synovial biopsy sections (3 �m) obtained from10 patients with RA were deparaffinized with xylene andrehydrated through a series of graded alcohols. Followingantigen retrieval (45 minutes at 95°C in Dako Target RetrievalSolution), slides were washed in Tris buffered saline (TBS;0.05M Tris, 0.15M NaCl, pH 7.5), incubated with 0.3% (v/v)hydrogen peroxide in methanol for 15 minutes, and treatedwith serum-free protein block (Dako) for 15 minutes at roomtemperature. Sections were then incubated for 1 hour with aTSG-6–specific polyclonal antiserum, RAH-1 (27) (1:800 inTBS), washed in TBS, and developed using a standardizedhorseradish peroxidase/diaminobenzidine/chromogen method(Dako REAL EnVision kit) according to the manufacturer’sinstructions. Slides were counterstained using Mayer’s hema-toxylin, dehydrated, cleared in xylene, and mounted using DPXprior to light microscopy. Expression of TSG-6 in the synoviumwas assigned a score of 0–4 according to the total number oflabeled cells (in the lining and sublining) per field of view at100� magnification (0 � no cells, 1 � 1–5 cells, 2 � 6–10 cells,
Figure 1. Osteoprotegerin (OPG) and TSG-6 act in synergy to inhibit soluble RANKL (sRANKL)–induced osteoclastic bone erosion. Humanperipheral blood mononuclear cells were cultured with sRANKL and macrophage colony-stimulating factor, in the absence or presence ofantiresorptive agents, for 21 days on dentine slices. a, The effects of OPG (0–10 nM) alone (n � 6) or combined with equivalent molar concentrationsof either recombinant human TSG-6 (n � 3) or Link_TSG6 (n � 3) were compared. b, The effects of equivalent molar concentrations of OPG (0–2.6nM) or TSG-6 (0–2.6 nM) were compared with those of half-molar equivalent concentrations of the 2 proteins combined (n � 12). Values are themean � SEM. � � P � 0.05; �� � P � 0.01; ��� � P � 0.001 versus OPG alone.
1036 MAHONEY ET AL
3 � 11–15 cells, and 4 � �16 cells). Sections were scored in ablinded manner by 2 independent observers. Normal rabbitserum was used as a control.
Detection of TSG-6 in SF. Synovial fluid samples werecentrifuged at 3,000g for 15 minutes at 4°C to remove anycellular components and digested with 2 mg/ml testicularhyaluronidase type IV-S (Sigma-Aldrich) for 1 hour at roomtemperature. SF was diluted 1:4 in PBS, and the concentra-tions of TSG-6 and OPG were determined by ELISA, asdescribed above.
Effect of TSG-6 on osteoclast formation/activationfrom SF macrophages. Macrophages were isolated as de-scribed previously (28) from the SF of patients with RA andseeded onto dentine slices (for lacunar resorption assays) orcoverslips (for TRAP assays) in 96-well plates at 1 � 106
cells/well. Cultures were maintained for 14 days with 25 ng/mlM-CSF and 50 ng/ml sRANKL, in the presence or absence of8.3 nM (250 ng/ml) OPG or 8.3 nM (457 ng/ml) rhTSG-6.Osteoclast formation was determined by the presence ofTRAP-positive multinucleated cells, and the extent of lacunarresorption (determined in quadruplicate for each individual)was measured as an indicator of osteoclast activity. Theseassays were also carried out for SF macrophages cultured inthe presence of TNF� (50 ng/ml) and M-CSF (25 ng/ml);TSG-6 concentrations in the culture media on days 3, 7, 10,and 14 were measured by ELISA, as described above.
Statistical analysis. Each set of experiments was re-peated at least 3 times. Differences between groups wereanalyzed using Student’s unpaired 1-tailed t-test. In each case,P values less than 0.05 were considered significant. Data fromthe measurement of resorption lacunae were normalized andexpressed as the percent relative to controls. Levels of TSG-6and OPG in SF samples from patients with arthritis werecompared with those in samples from nondisease controls,using a nonparametric Mann-Whitney test. Regression analy-ses were performed with a least squares (linear) method usingKaleidograph 4.0 (Synergy Software).
RESULTS
TSG-6 acts in synergy with OPG. We reportedpreviously that the potencies of TSG-6 and OPG toinhibit lacunar resorption by PBMC-derived osteoclastswere similar; i.e., median inhibition concentration (IC50)values of �0.5 nM and �0.25 nM, respectively (5). Here,we compared the effects of OPG (0–10 nM) alone and incombination with equivalent molar concentrations ofeither rhTSG-6 or Link_TSG6. Although the presenceof rhTSG-6 gave rise to a significant increase in theantiresorptive activity of OPG at low doses (e.g., �4-foldat the 0.02-nM dose), Link_TSG6 had no such effect(Figure 1a). In additional experiments comparing theactivities of equivalent molar concentrations of OPGand rhTSG-6 administered alone with those of thecorresponding half-molar concentration of each reagentin combination (i.e., comparing equivalent total concen-trations of protein), we again observed that rhTSG-6
Figure 2. Cytokine-induced expression of TSG-6 during osteoclastogen-esis in vitro. a, Human peripheral blood mononuclear cells (PBMCs)were cultured with macrophage colony-stimulating factor (M-CSF)and 50 ng/ml of pro-osteoclastogenic agents for 21 days, at which timeTSG-6 and osteoprotegerin (OPG) protein production was assessed byenzyme-linked immunosorbent assay (ELISA). b, PBMCs were cul-tured with M-CSF and 0, 1, 5, 10, or 50 ng/ml of proinflammatorycytokines, and TSG-6 production was determined by ELISA. Valuesare the mean � SEM (n � 4). � � P � 0.05; �� � P � 0.01;��� � P � 0.001 versus unstimulated cells. CTRL � control; IL-1 �interleukin-1; sRANKL � soluble RANKL; TNF� � tumor necrosisfactor �.
TSG-6 IS AN AUTOCRINE REGULATOR OF BONE RESORPTION 1037
significantly enhanced the antiresorptive effect of OPG(Figure 1b). Overall, the data presented in Figure 1demonstrated that, although OPG alone inhibited den-tine erosion with an IC50 value of �0.25 nM, the IC50value for OPG plus rhTSG-6 was �0.02 nM. This�10-fold reduction in the IC50 value indicated that OPGand TSG-6 act in synergy to inhibit osteoclastic boneresorption.
Inflammatory cytokines induce TSG-6 secretionby osteoclasts and their precursors. We previouslyshowed that osteoclasts derived from the long bones ofTSG-6�/� mice are more resorptive than those fromwild-type controls, indicating that TSG-6 expressed bycells of an osteoclast lineage might act in an autocrinemanner to inhibit osteoclast activation (5). Here, weinvestigated whether TSG-6 is expressed in response tofactors that have been reported to promote osteoclasto-genesis but have differing effects on bone resorption(29–33). Although IL-1, IL-6, TNF�, IL-17, LIGHT,and sRANKL all induced polykaryon formation fromPBMCs, as confirmed by TRAP-positive staining (datanot shown), after 21 days of culture TSG-6 was detectedonly in supernatants of cells stimulated with TNF�, IL-1,or IL-6; OPG was not secreted in response to any of thefactors tested (Figure 2a).
ELISA of culture supernatants collected every3–4 days during the 21-day period associated withosteoclast formation/activation revealed that TSG-6 se-cretion was dose dependent for each cytokine tested,with the highest levels being expressed in response toTNF� (Figure 2b). For cells treated with TNF� (5, 10,and 50 ng/ml), TSG-6 secretion increased up to day 14(i.e., during osteoclast formation) and then tended toplateau before increasing again during days 17–21. Thus,mature osteoclasts produced substantially more TSG-6than did osteoclast precursors in response to a givendose of TNF�. Similar effects were observed for cellscultured with IL-6. In contrast, cells treated with IL-1showed a rapid rise in TSG-6 production between days 7and 10, but then production reached a plateau.
Induction of TSG-6 expression by inflammatorycytokines correlates with inhibition of lacunar resorp-tion. PBMCs cultured on dentine slices were induced toundergo osteoclast formation/activation by sRANKLand M-CSF in the presence of TNF�, IL-1, or IL-6 (0–75ng/ml) (Figure 3a). The lowest level of lacunar resorp-tion (after 21 days) was observed for cells cultured with75 ng/ml of TNF� (�30% resorption compared with theno-cytokine control, which was defined as 100% resorp-tion). IL-1 and IL-6 were less effective inhibitors at thisdose (�60% resorption), although at the lower doses
Figure 3. Secretion of TSG-6 during osteoclastogenesis correlateswith an inhibition of RANKL-mediated lacunar resorption. a and b,Human PBMCs were cultured in the presence of sRANKL and M-CSFfor 21 days on dentine slices or coverslips. The addition of TNF� (●),IL-1 (�), and IL-6 (f) to these cultures caused a dose-dependentreduction in lacunar resorption (a), which correlated (R � 0.82) withTSG-6 levels as determined by ELISA (b). Values in a are the mean �SEM (n � 3). � � P � 0.05; ��� � P � 0.001 versus cells cultured withsRANKL alone. Data in b are from cells treated with 5, 25, and 75ng/ml of cytokine (n � 3). c, Supplementation with TNF� (75 ng/ml)had no effect on cell viability after 21 days, as determined using aLIVE/DEAD assay kit, where live cells are stained green and deadcells are stained red; cell nuclei are visualized in blue. Bars � 100 �m.See Figure 2 for definitions.
1038 MAHONEY ET AL
tested (1, 5 and 25 ng/ml), all 3 cytokines had similareffects (Figure 3a).
The addition of cytokines had no apparent effecton cell viability, as illustrated for TNF� (Figure 3c),indicating that cell death was not the cause of reducedresorption. Because exogenously added rhTSG-6(�0.1–10 nM) (see Figure 1) inhibited sRANKL-induced osteoclastic resorption in vitro, we consideredthe possibility that this cytokine-mediated inhibition oflacunar resorption might result from up-regulated ex-pression of TSG-6. Consistent with this, sRANKL alonedid not induce TSG-6 (Figure 2a), but the amount ofTSG-6 secreted by mature osteoclasts in the presence of50 ng/ml of TNF�, IL-1, and IL-6 (i.e., �70 ng/ml [�2nM], �15 ng/ml [�0.4 nM], and �10 ng/ml [�0.3 nM] ofTSG-6, respectively) (Figure 2b) was within the 0.1–10nM range. In this regard, the measurement of TSG-6protein (on day 21) in the supernatants from resorptionassays performed in the presence of TNF�, IL-1, or IL-6revealed a positive correlation between the ability ofthese cytokines to induce TSG-6 expression and theextent to which they inhibit lacunar resorption (Figure3b).
TSG-6 inhibits lacunar resorption by an auto-crine mechanism. To further investigate whether TSG-6acts in an autocrine manner to inhibit bone resorption inthe presence of inflammatory cytokines, a TSG-6–
specific monoclonal antibody (A38.1) (25) was added tosRANKL-stimulated osteoclast cultures supplementedwith rhTSG-6 or TNF�. This antibody effectively abol-ished the antiresorptive effect of rhTSG-6 (Figure 4a)and significantly suppressed the inhibition of resorptioninduced by TNF� (Figure 4b). PBMCs cultured for 21days with TNF� alone (without sRANKL or M-CSF) didnot form bone-resorbing osteoclasts, and this was notaffected by A38.1 (Figure 4b). These data suggest thatTSG-6 produced by osteoclasts is responsible, at least inpart, for the reduction in lacunar resorption mediated byTNF�. Furthermore, PBMCs cultured for 21 days withsRANKL/M-CSF formed F-actin rings, a characteristicof osteoclasts that are immobilized on bone and arequirement for resorptive activity. As seen in Figure 4c,this formation was substantially reduced by the additionof rhTSG-6.
Overall, the results described above indicate thatTSG-6 is expressed by mature osteoclasts in response toinflammatory cytokines and acts to inhibit osteoclastactivation in an autocrine manner.
Expression of TSG-6 in the inflamed joints ofpatients with arthritis. Immunostaining of tissue frompatients with RA revealed the presence of TSG-6 in thesynovium (Figure 5a) and pannus, which is consistentwith previous observations (9). Immunofluorescencestaining demonstrated that TSG-6 was localized to a
Figure 4. TSG-6 inhibits osteoclast activation in response to the proinflammatorycytokine TNF� through an autocrine mechanism. a and b, Human PBMCs werecultured with sRANKL and M-CSF, in the absence or presence of TSG-6 (250ng/ml), TNF� (50 ng/ml), and A38.1, a TSG-6–specific monoclonal antibody (1�g/ml), for 21 days on dentine slices. A38.1 completely abolished the inhibitory effectof TSG-6 on osteoclast resorption (a) and partially suppressed the antiresorptiveeffect of TNF� (b); A38.1 had no effect on cells cultured with sRANKL/M-CSF (a)or TNF� (b) alone. Values are the mean � SEM (n � 3). ��� � P � 0.001. c, Stainingof multinucleated cells cultured in the presence of TSG-6 with tetramethylrhodamineisothiocyanate–conjugated phalloidin revealed a substantial reduction in filamentousactin ring formation. Bars � 50 �m. R � sRANKL M-CSF; T � TNF�; Ab �A38.1; NS � not significant (see Figure 2 for other definitions).
TSG-6 IS AN AUTOCRINE REGULATOR OF BONE RESORPTION 1039
subset of cells that are distinct from CD68 macro-phages (data not shown). ELISAs showed that TSG-6,like OPG, was present in the clarified SF of patients withvarious arthropathies (Figure 5b). Here, we observed apositive correlation (R � 0.81) between the level ofTSG-6 in SF and the TSG-6 immunoreactivity of syno-vial tissue from the same patients with RA (Figure 5a).Both TSG-6 and OPG were present at a level of �1ng/ml in noninflammatory, nonosteolytic control SF, butthe levels of TSG-6 and OPG were substantially elevatedin all of the arthritides investigated. Considerable vari-ability in protein levels was evident both between differ-ent clinical disorders and among patients with a givencondition. As shown in Figure 5b, expression of TSG-6was significantly elevated in patients with gout, patientswith RA, patients with inflammatory OA, and patientswith PPA.
TSG-6 inhibits the activation but not the for-mation of osteoclasts from SF macrophages. Wepreviously reported that macrophages derived fromthe SF of patients with arthritis undergo osteoclasto-genesis and mediate lacunar resorption in response to
sRANKL (28). Here, we showed that rhTSG-6, unlikeOPG, had no effect on sRANKL-induced differentia-tion of macrophages from patients with RA (Figure6a). However, in common with OPG, rhTSG-6 signif-icantly inhibited lacunar resorption by these cells(Figure 6b), which is consistent with our previous datafrom PBMCs (5). When SF macrophages were cul-tured in the presence of TNF�, osteoclast formationwas similar to that induced by sRANKL (Figure 6a),but there was little or no osteoclastic resorption(Figure 6b). TSG-6 was detected in the media of SFmacrophages cultured in the presence of TNF� (Fig-ure 6c). Overall, these observations suggest that theexposure of osteoclast precursors to TNF� inducesTSG-6 expression, which may operate locally to in-hibit bone erosion by blocking osteoclast activationduring inflammation in vivo. Consistent with thesefindings, immunohistochemical analysis of thecartilage–bone interface in patients with RA revealedexpression of TSG-6 by giant multinucleated cellsadjacent to resorption lacunae (Figure 6d).
Figure 5. Quantification of TSG-6 protein in arthritic joints. a, Immunohistochemical analysis of synovial tissue from patients with rheumatoidarthritis (RA) using the polyclonal antiserum RAH-1 showed localization of TSG-6 within the lining and sublining, the extent of which correlatedwith TSG-6 levels in paired synovial fluid (SF) samples (R � 0.81). Values are the mean � SEM. Bar � 50 �m. b, TSG-6 and osteoprotegerin (OPG)in clarified SF samples from 11 patients with noninflammatory osteoarthritis (NON-INFL OA), 8 patients with inflammatory (INFL) OA, 34patients with RA, 7 patients with psoriatic arthritis (PA), 9 patients with gout, and 13 patients with pyrophosphate arthritis (PPA), and from 3nondisease control subjects (CTRL) were quantified by enzyme-linked immunosorbent assay. Data are presented as box plots, where the boxesrepresent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90thpercentiles. � � P � 0.05; �� � P � 0.01; ��� � P � 0.001 versus control. AU � arbitrary units; NS � not significant.
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DISCUSSION
In this study, we showed that TSG-6 is expressedby osteoclasts (and their precursors) in response toproinflammatory cytokines and can inhibit osteoclastactivation, by impairment of F-actin ring formation, inan autocrine manner. Importantly, we demonstratedthat TSG-6 and the well-established antiresorptive pro-tein OPG work in synergy to block osteoclastic boneerosion.
OPG acts by directly inhibiting the binding ofRANKL to RANK on the surface of osteoclasts, whichprevents triggering of both the canonical and noncanoni-cal NF-�B signaling pathways (for review, see ref. 34).Although TSG-6, like OPG, has been shown to bindRANKL (5), how this interaction contributes to theantiresorptive effect of TSG-6 and which signaling path-ways are involved remain to be determined. However,the finding that TSG-6 and OPG act synergisticallysuggests that they mediate their antiresorptive effectsthrough different mechanisms. Consistent with this,OPG reduces bone resorption by inhibiting osteoclastdifferentiation (30,35) (Figure 6a), activation (35,36)(Figure 6b), and cell survival (37), whereas TSG-6
specifically down-regulates the resorptive activity ofmature osteoclasts (Figures 1, 3, and 6b), with no impacton osteoclast formation from either PBMCs (5) or SFmacrophages (Figure 6a).
Neither osteoclasts (Figure 2a) nor their precur-sors express OPG in response to various inflammationmediators. However, our data indicate that the inflam-matory cytokines TNF�, IL-1, and IL-6 promote secre-tion of TSG-6 by these cells, and that there is an inversecorrelation between the TSG-6 concentration and oste-oclastic resorption. The observation that TSG-6 inhibitsF-actin ring formation suggests a novel autocrine mech-anism by which osteoclasts produce TSG-6 in responseto inflammatory cytokines and thereby limit their ownability to anchor to and resorb bone; that is, osteoclas-togenesis is uncoupled from activation. Nonetheless, wecannot exclude the possibility that TSG-6 produced byother cell types also regulates osteoclast function (e.g.,in a paracrine manner).
Inflammatory cytokines are abundant in condi-tions such as RA and have been implicated in osteoclas-tic bone erosion. TNF� and IL-1 can be indirectlyosteoclastogenic by promoting the expression of
Figure 6. TSG-6 inhibits the activation of osteoclasts derived from synovial fluid (SF) macrophages and is expressed by cells at the synovium–boneinterface in patients with rheumatoid arthritis (RA). SF cells from 4 patients with RA were cultured with macrophage colony-stimulating factor(M-CSF) in the presence of soluble RANKL (with or without TSG-6 [8.3 nM] or osteoprotegerin [OPG; 8.3 nM]) or with M-CSF in the presenceof tumor necrosis factor � (TNF�; 50 ng/ml) for 14 days on coverslips or dentine slices. a–c, Osteoclast formation was analyzed by tartrate-resistantacid phosphatase staining (a), and osteoclast activation was determined by lacunar resorption (b). TSG-6 secreted during TNF�-drivenosteoclastogenesis was quantified by enzyme-linked immunosorbent assay (c). Values are the mean � SEM (n � 4). � � P � 0.05; �� � P � 0.01versus cells cultured in the absence of TNF�; ��� � P � 0.001 versus M-CSF plus RANKL alone. d, Expression of TSG-6 by osteoclasts (‚) at thesynovium–bone interface (Œ) of patients with RA was visualized by immunohistochemistry; one representative example of 4 is shown. Bar � 100 �m.R � M-CSF sRANKL; T � M-CSF TNF�; CTRL � control.
TSG-6 IS AN AUTOCRINE REGULATOR OF BONE RESORPTION 1041
RANKL by osteoblasts (38) and synovial fibroblasts(39), while IL-1 (40), IL-6 (32), and TNF� (31,41,42)have been reported to stimulate osteoclast formationand induce resorptive activity directly. Furthermore,there is evidence that TNF� and IL-1 secreted byosteoclast precursors might act via autocrine mecha-nisms to enhance osteoclast formation during inflamma-tion (for review, see ref. 43). The binding of TNF� to itsreceptor on osteoclasts (and their precursors) activatesthe same NF-�B signaling pathways as those activated byRANKL. However, a recent study showed that, unlikeRANKL, TNF� promotes the accumulation of NF-�Bp100 in osteoclast precursors, which acts as a suppressorof the noncanonical NF-�B pathway (44). This limitingeffect of TNF� on osteoclastogenesis was shown toprotect against bone erosion and inflammation in amodel of RA in mice null for NF-�B2 (34,44).
Our data suggest another mechanism wherebyTNF� and other inflammatory cytokines might limitbone erosion during inflammation, i.e., by inducingsecretion of TSG-6 by osteoclasts, leading to autocrineinhibition of osteoclast activation. The finding that theantiresorptive effect of TNF� was only partially inhib-ited by an anti–TSG-6 monoclonal antibody (whereasthis monoclonal antibody completely ablated the effectof rhTSG-6 on RANKL-induced resorption) (Figure 4)indicates that TNF� also promotes proresorptive mech-anisms that are not inhibited by TSG-6. The balancebetween the proresorptive and antiresorptive effects ofTNF� (where the outcome in vivo would likely dependon the relative concentrations of the many factors thatcontribute to osteoclast formation/activation) might helpto explain why some patients receiving anti-TNF therapycontinue to experience bone erosion despite apparentclinical remission, while others experience a reduction injoint destruction in spite of ongoing disease activity (45).
In conclusion, our observation that TSG-6 pro-duced by osteoclasts in response to certain inflammatorycytokines (i.e., TNF�, IL-1, and IL-6) can inhibit boneerosion, coupled with the antiinflammatory and chondro-protective effects of this protein (13–17), indicate a uniquerole for TSG-6 as an endogenous protector of joint tissuesduring inflammation. Although in the complex milieu ofthe inflamed joint, TSG-6 may serve only to diminishrather than abolish joint damage, the use of such a natu-rally occurring protective mechanism may represent animportant therapeutic option for the osteolytic joint.
ACKNOWLEDGMENTSWe thank Marilyn Rugg (Medical Research Council
Immunochemistry Unit, Oxford) and Dacha Tongsoongnoen
(University of Manchester) for the production of rhTSG-6 andLink_TSG6, Dr. Kay Chapman (Nuffield Department of Or-thopaedics, Rheumatology and Musculoskeletal Sciences[NDORMS]) for advice on statistical analysis, and Dr. ZhidaoXia (NDORMS) for advice on microscopy. Research at theNuffield Department of Orthopaedics, Rheumatology andMusculoskeletal Sciences, Oxford University is supported bythe National Institute for Health Research (UK), BiomedicalResearch Unit into Musculoskeletal Disease.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Sabokbar had full access to all ofthe data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Study conception and design. Mahoney, Swales, Athanasou, Pitzalis,Sharif, Day, Milner, Sabokbar.Acquisition of data. Mahoney, Swales, Athanasou, Bombardieri,Kliskey, Sharif.Analysis and interpretation of data. Mahoney, Swales, Bombardieri,Sharif, Day, Milner, Sabokbar.
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